Fundamentals%20of%20modern%20UV-visible%20spectroscopy

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Fundamentals of modern UV-visible spectroscopy

• Presentation Materials

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The Electromagnetic Spectrum
n c / l
E hn
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Electronic Transitions in Formaldehyde
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Electronic Transitions and Spectra of Atoms
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Electronic Transitions and UV-visible Spectra in
Molecules
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Derivative Spectra of a Gaussian Absorbance Band
Absorbance
1st Derivative
2nd Derivative
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Resolution Enhancement

• Overlay of 2
• Gaussian bands
• with a NBW of 40 nm separated
• by 30 nm

• Separated by
• 4th derivative

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Transmission and Color
The human eye sees the complementary color to
that which is absorbed
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Absorbance and Complementary Colors
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Transmittance and Concentration The
Bouguer-Lambert Law
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Transmittance and Path LengthBeers Law
Concentration
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The Beer-Bouguer-Lambert Law
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Two-Component Mixture
Example of a two-component mixture with little
spectral overlap
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Two-Component Mixture
Example of a two-component mixture with
significant spectral overlap
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Influence of 10 Random Error

• Influence on the calculated concentrations
• Little spectral overlap 10 Error
• Significant spectral overlap Depends on
  similarity, can be much higher (e.g. 100)

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Absorption Spectra of Hemoglobin Derivatives
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Intensity Spectrum of the Deuterium Arc Lamp

• Good intensity
• in UV range
• Useful intensity
• in visible range
• Low noise
• Intensity decreases
• over lifetime

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Intensity Spectrum of the Tungsten-Halogen Lamp

• Weak intensity in
• UV range
• Good intensity in
• visible range
• Very low noise
• Low drift

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Intensity Spectrum of the Xenon Lamp

• High intensity in UV range
• High intensity in visible range
• Medium noise

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Dispersion Devices

• Non-linear dispersion
• Temperature sensitive

• Linear Dispersion
• Different orders

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Photomultiplier Tube Detector

• High sensitivity at
• low light levels
• Cathode material
• determines spectral sensitivity
• Good signal/noise
• Shock sensitive

Anode
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The Photodiode Detector

• Wide dynamic range
• Very good
• signal/noise at high light levels
• Solid-state device

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Schematic Diagram of a Photodiode Array

• Same characteristics
• as photodiodes
• Solid-state device
• Fast read-out cycles

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Conventional Spectrophotometer
Schematic of a conventional single-beam
spectrophotometer
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Diode-Array Spectrophotometer
Schematic of a diode-array spectrophotometer
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Diode-Array Spectrophotometer
Optical diagram of the HP 8453 diode-array
spectrophotometer
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Conventional Spectrophotometer
Optical system of a double-beam spectrophotometer
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Diode-Array Spectrophotometer
Optical system of the HP 8450A diode-array
spectrophotometer
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Conventional Spectrophotometer
Optical system of a split-beam spectrophotometer
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Definition of Resolution
Spectral resolution is a measure of the ability
of an instrument to differentiate between two
adjacent wavelengths
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Instrumental Spectral Bandwidth
The SBW is defined as the width, at half the
maximum intensity, of the band of light leaving
the monochromator
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Natural Spectral Bandwidth
The NBW is the width of the sample absorption
band at half the absorption maximum
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Effect of SBW on Band Shape
The SBW/NBW ratio should be 0.1 or better to
yield an absorbance measurement with an accuracy
of 99.5 or better
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Effect of Digital Sampling
The sampling interval used to digitize the
spectrum for computer evaluation and storage also
effects resolution
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Wavelength Resettability
Influence of wavelength resettability on
measurements at the maximum and slope of an
absorption band
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Effect of Stray Light
Effect of various levels of stray light on
measured absorbance compared with actual
absorbance
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Theoretical Absorbance Error
The total error at any absorbance is the sum of
the errors due to stray light and noise (photon
noise and electronic noise)
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Effect of Drift
Drift is a potential cause of photometric error
and results from variations between the
measurement of I0 and I
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Transmission Characteristics of Cell Materials
Note that all materials exhibit at least
approximately 10 loss in transmittance at all
wavelengths
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Cell Types I
Open-topped rectangular standard cell (a) and
apertured cell (b) for limited sample volume
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Cell Types II
Micro cell (a) for very small volumes and
flow-through cell (b) for automated applications
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Effect of Refractive Index
Changes in the refractive index of reference and
sample measurement can cause wrong absorbance
measurements
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Non-planar Sample Geometry
Some sample can act as an active optical
component in the system and deviate or defocus
the light beam
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Effect of Integration Time
Averaging of data points reduces noise by the
square root of the number of points averaged
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Effect of Wavelength Averaging

• Wavelength averaging reduces also the noise
  (square root of data points)
• Amplitude of the signal is affected

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Increasing Dynamic Range
Selection of a wavelength in the slope of a
absorption band can increase the dynamic range
and avoid sample preparation like dilution
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Scattering
Scattering causes an apparent absorbance because
less light reaches the detector
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Scatter Spectra

• Rayleigh scattering Particles small relative to
  wavelength
• Tyndall scattering Particles large relative to
  wavelength

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Isoabsorbance Corrections
Absorbance at the reference wavelength must be
equivalent to the interference at the analytical
wavelength
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Background Modeling
Background modeling can be done if the
interference is due to a physical process
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Internal Referencing
Corrects for constant background absorbance over
a range
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Three-Point Correction

• Uses two reference wavelengths
• Corrects for sloped linear background absorbance

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Discrimination of Broad Bands

• Derivatives can eliminate background absorption
• Derivatives discriminate against broad
  absorbance bands

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Scatter Correction by Derivative Spectroscopy
Scatter is discriminated like a broad-band
absorbance band
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Effect of Fluorescence
The emitted light of a fluorescing sample causes
an error in the absorbance measurement
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Acceptance Angles and Magnitude of Fluorescence
Error

• Forward optics Absorbance at the excitation
  wavelengths are too low
• Reversed optics Absorbance at the emission
  wavelengths are too low

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Inadequate Calibration

• Theoretically only one standard is required to
  calibrate
• In practice, deviations from Beers law can
  cause wrong results

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Calibration Data Sets

• Forward optics Absorbance at the excitation
  wavelengths are too low
• Reversed optics Absorbance at the emission
  wavelengths are too low

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Wavelength(s) for Best Linearity

• A linear calibration curve is calculated at each
  wavelength
• The correlation coefficient gives an estimate on
  the linearity

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Wavelength(s) for Best Accuracy

• The quantification results are calculated at
  each wavelength
• The calculated concentration are giving an
  estimate of the accuracy

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Precision of an Analysis
Precision of a method is the degree of agreement
among individual test results when the procedure
is applied repeatedly to multiple samplings
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Wavelength(s) for Best Sensitivity

• Calculation of relative standard deviation of
  the measured values at each wavelength
• The wavelength with lowest RSD likely will
  yield the best sensitivity

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Wavelength(s) for Best Selectivity
Selectivity is the ability of a method to
quantify accurately and specifically the analyte
or analytes in the presence of other compounds
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Ideal Absorbance and Wavelength Standards

• An ideal absorbance standard would have a
  constant absorbance at
• all wavelengths
• An ideal wavelength standard would have very
  narrow, well-defined
• peaks

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Ideal Stray Light Filter
An ideal stray light filter would transmit all
wavelengths except the wavelength used to measure
the stray light
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Holmium Perchlorate Solution
The most common wavelength accuracy standard is a
holmium perchlorate solution
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Potassium Dichromate Solution
The photometric accuracy standard required by
several pharmacopoeias is a potassium dichromate
solution
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Stray Light Standard Solutions
The most common stray light standard and the
respectively used wavelengths
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Toluene in Hexane (0.02 v/v)
The resolution is estimated by taking the ratio
of the absorbance of the maximum near 269 nm and
minimum near 266 nm
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Confirmation Analysis
In confirmation analysis, the absorbance at one
or more additional wavelengths are used to
quantify a sample
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Spectral Similarity
Comparative plots of similar and dissimilar
spectra
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Precision and Accuracy
Precision
Precision
Precision
Precision
Accuracy
Accuracy
Accuracy
Accuracy
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Hydrolysis of Sultone
Absorbance AU
Wavelength nm